tnt. Archs Allergy appl. Immun. 60: 377-382 (1979)
Interferon Enhanced Human Natural Killer and AntibodyDependent Cell-Mediated Cytotoxic Activity A. M. AItallah and T. Folks Division of Pathology, Bureau of Biologies, FDA, and Department of Immunology, Naval Medical Research Institute, Bethesda, Md.
Introduction Natural killer (NK) or spontaneous cyto toxicity has been well studied, recognized and reviewed in man and other species [5, 9, 12, 18]. The absence of apparent antigen ic stimulation is the hallmark of this phe nomenon, indicating that specificity may not be important in immunosurveillance against tumors. Separation and identification of the effector cells which mediate NK activity and antibody-directed cell-mediated cytotoxicity (ADCC) have recently been attempted [8, 11, J3]. These studies suggest that the two
phenomena are mediated by different effec tor cell subpopulations and also by different mechanisms [11, 16]. Since the finding that some strains of mice showing high NK activity have low spontaneous tumor development [9], the search for NK enhancing products for possi ble cancer immunotherapy has received a great deal of attention [3], One promising candidate is interferon. Virally infected cells induce high levels of human interferon (HIF) from human peripheral blood lym phocytes (HPBLs) when cultured together [15, 17], The HPBLs show enhanced NK
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
Abstract. Preincubation of normal human lymphocytes with human interferon for 18-24 h at 37 °C resulted in an increase of the activity of both natural killer (NK) cells and anti body-mediated cytotoxic cells (ADCC). The human myeloid line, K-562, which is highly susceptible to NK cells, was employed. ADCC was assessed with antibody-coated chick erythrocytes as targets. NK cells and ADCC were detected in a 4-hour 5lCr release assay. The magnitude of the enhancement was proportionate to the amount of interferon used in preincubation of the effector cells. Prcincubation of tumor-target cells with interferon does not increase their susceptibility or resistance to lysis. The major effect of interferon on the cellular metabolism of the tumor-target cell is inhibition of DNA synthesis, but no direct cytotoxic effect was detected. Our findings may be important in understanding the mode of action of interferon in increasing host resistance to a variety of pathogens and tumors. This may be accomplished by inhibiting the growth of the tumor while simultaneously enhancing the natural killing mechanism for immunosurveillance.
Attallah Folks
activity toward these infected cells and are Fc-receptor-positivc and Ig-negative [6]. Some reports using human fibroblasts with anti-HLA or antivirus sera, and virus-in fected cells, have shown no enhancement of ADCC with HPBLs [15, 17]. The possibili ty has been raised that HIF may be acting not only on the effector cell, but also on the tumor cell. In the study reported here, HPBLs and tumor target cells were incubat ed with HIF and tested for enhanced NK and ADCC activities.
Materials and Methods
at the beginning to enhance effector and target cell contact. Plates were incubated for 4 h at 37 C in 5"/ C 0 2. At the end of incubation, the plates were spun at 500 g for 10 min, and 0.1 ml of the 0.2 ml total volume was counted for ra dioactivity (1185 automatic gamma counter, Searle, Chicago, 111.). ADCC Assay HPBLs (5XlOVlOO/d) were added to chrom ium-labeled CRBC (2X 10V50 «1) in triplicate in Linbro 96-well roundbottom microtiter trays. In addition, either 50 /d of anti-CRBC (1:20,000 fi nal dilution) or 50 «1 medium was added to the wells (total volume was 200 id). Plates were incu bated for 4 h at 37 C in a 5°/o C 0 2 incubator, at which time the plates were spun and 0.1 ml from each well was counted for radioactivity.
Effector Cells Human peripheral blood, which demonstrated high NK cell activity for all experiments, was ob tained from normal donors. Heparinized peripher al blood was layered on Ficoll-Hypaque and cen trifuged for 40 min at 500 g at 4 C. Mononuclear cells were removed and washed three times in RPMI-1640.
Interferon Source and Treatment Partially purified human lymphoblastoid inter feron induced in Namalva cells with a specific ac tivity of 1.25 X105 reference units/mg protein was used [1], HPBLs, 2X l06/ml RPMI-1640, were in cubated with HIF for 24 h at 37 °C in a 5°/o C 0 2 incubator. Cells were washed three times before use in the assay.
Target Cells For the NK assay, the myeloid leukemia cell, K-562, was utilized. It was grown in culture using complete RPMI-1640 media supplemented with 250 ug/ml gentamycin, 10 miM Hepes, 2 mM glu tamine and 10°/o heat-inactivated fetal bovine se rum (FBS) until time of the assay. 2X106 tumor cells were washed twice in RPMI-1640 and then resuspended in 100 «Ci/100 «1 of 5,Cr (Amersham). The tumor cells were incubated for 1 h at 37 °C in 5°/o C 0 2 and washed three times in RPMI-1640. Chicken red blood cells (CRBC) were freshly obtained for each ADCC assay, washed twice in complete RPMI-1640, and treated in the same way as the tumor cells.
Calculation of Percent Cytotoxicity Percent cytotoxicity using 5lCr release assay was determined by the following formula:
NK Assay HPBLs (5X105/100 «1) were incubated with chromium-labeled K-562 cells (1X104/100 m1) in triplicate in Linbro 96-well roundbottom micro titer trays. Medium was always complete RPMI1640. A slow spin (50 g for 1 min) was employed
% cytotoxicity = test cpm - spontaneous release cpm 100 x ---------------------------------------------------- • treeze/thaw cpm - spontaneous release cpm DNA, RNA and Protein Synthesis in the Target cells The myeloid leukemia cell, K-562, was sus pended to lX 106/ml in 0.1ml and dispersed in each well of a microtiter plate (Linbro Corp., New Haven, Conn.) in triplicate. Various doses of interferon (0.1 ml) were added to the culture. The cultures were then incubated at 37 °C in a humidi fied atmosphere of 5°/o C 0 2 in air for 48 h. Each culture received either 1 «Ci of 3H-TdR, 3H-uridine or 3H-leucine (Schwarz-Mann, Orangeburg, N.Y.) in 20 »1 of medium 16 h before harvest. Cells were harvested using the multiple automated sample harvester. The mean uptake and standard
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
378
Enhanced Human NK and ADCC Activity by HIF
error were calculated; percent inhibition was cal culated by the formula: % inhibition = mean cpm of culture containing HIF x 100. 1 mean cpm of culture containing medium
Results Effect of HIF on NK Activity Variation of Effector-to-Target Ratios. In order to determine the effect of interfer70 60
t 50 O 30
20 10 -
25:1
10:1
5:1
E :T RATIO
Fig. 1. Enhancement of NK activity by human interferon. HPBLs were incubated for 24 h with 5,000 U HIF ( • ) or for 24 h with medium alone (O). HPBLs (effector) were incubated with 51Crlabeled K-562 (target) for 4 h, and net percent NK cytotoxicity was determined. Vertical bars = + S.E.
70 5
O O
on on HBPLs, the effector-to-target (E:T) ratios were varied from 25:1 to 5:1, as seen in figure I. At all ratios, significant en hancement of NK activity was observed with the HIF-treated cells. The greatest enhance ment was seen at the 10:1 E:T ratio, with the HIF-treated cells having 50 ± 2%> cyto toxic activity, while the untreated cells showed 31 ± 2°/o. Dose Response with Varying Units of Interferon. Figure 2 depicts the effect of varying the units of interferon on NK activ ity. Maximal enhancement was observed when 600 U HIF were added to HPBLs and incubated for a 24-hour period. There was no further increase in cytotoxicity when HPBLs were treated with increasing doses of HIF, up to 5,000 U. Interferon Treatment of Target Cells. In order to determine whether the effect of HIF was on the effector or the target cell, K-562 cells were incubated with 5,000 U of HIF and tested 24 h later for a change in susceptibility to lysis by HPBLs (table I). No change in susceptibility occurred follow ing target treatment with HIF (table I). However, as little as 5 U HIF caused signif icant inhibition of DNA synthesis. RNA and protein synthesis was also inhibited but to a lesser extent in the tumor target cells (table II). Table I. Effect of human interferon on the suscep tibility of the target cell in the NK assay1
60 50 40
0
125 300 600 1,500 2p00 5p00 U N ITS OF INTERFERON
Fig. 2. Effect of varying unit doses of interfer on on NK activity. Condition was as in figure 1; E:T ratio was 50:1. Vertical bars = + S.E.
Target
Effector
% cytotoxicity ± S.E
K-562 + HIF (5,000 U) K-562
+ HPBLs + HPBLs
58 ± 2 64 ± 3
1 Target cells (K-562) were incubated for 24 h in either medium + 5,000 U of HIF or medium alone.
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o
379
380
Tabic II. Effect of human interferon on DNA, RNA and protein synthesis of myeloid leukemia cells in vitro
HIF units per culture
% inhibition of 3H-TdR
0 5 50 500
3H-uridine 3H-leucine
_
_
_
32 37 53
2 24 30
15 7 22
Table III. Enhancement of ADCC following pre incubation of HIF with HPBLs1
Anti-CRBC
% ADCC ± S.E
1
1
+
1 +
+
1 +
Interferon
-
55 ± 1.5 26 ± 0.15 3.0 ± 0.1 0.1 ± 0.1
1 HPBLs were incubated either with or without HIF (5,000 U) for 24 h and tested for their ability to mediate ADCC against CRBC. E:T ratio =25:1. Final dilution of rabbit anti-CRBC was 1:20,000.
Enhancement in ADCC Activity Follow ing Interferon Treatment of HPBLs. HPBLs were either treated for 24 h with HIF (5,000 U) or not treated and then test ed for their ability to mediate ADCC against CRBC. As observed in table III, a 2-fold increase in ADCC was seen in the HIFtreated effector population, with no signifi cant increase in cytotoxicity between HIFtreated and untreated effector groups in the absence of anti-CRBC.
Discussion
The present study has investigated some of the effects of HIF on HPBLs, specifically
NK and ADCC activity. It was found that NK activity of HPBLs was enhanced ap proximately 70"/o following treatment with HIF [2], Kinetics of the NK response stud ied by varying the effector-to-target ratios indicated that either more killer cells were present or each killer cell was more effec tive, since enhancement was greater at low er ratios. When units of HIF were varied, a maximal concentration was seen at 600 U after which there was no further enhance ment with increasing doses; this could be due to saturation of the HIF receptor. In order to rule out HIF carry-over from preincubation with HPBLs and the subse quent direct effect on the target cell, investi gations were carried out to determine if sus ceptibility to lysis of the target had been al tered. Since no significant change in suscep tibility was noted, it was concluded that the NK activity was contributed solely by the effector cell. However, HIF did affect pri marily DNA; RNA and protein synthesis were affected to a lesser extent in the tumor target. Reports from various laboratories have indicated that ADCC against virus-infected cells is not enhanced over normal cells [15] and that ADCC is not enhanced in the pres ence of HPBLs which were preincubated with HIF [17], Conflicting reports have shown that HIF can enhance ADCC using tumor cell targets [7], Differences in the two systems may reside in the source of effector cells, the antitumor antibody, or the quanti ty and source of HIF. In either case, our findings [2] have shown a marked increase in ADCC (greater than 2-fold). The type of target used in our system was xenogeneic, which suggests that another type of effector cell mediates ADCC against xenogeneic tar gets. Such a subpopulation of ADCC effec
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
Altai Iah/Folks
tor cells has recently been described in the murine system [14]. Further studies are being carried out to determine if there arc subpopulations of human ADCC effector cells and if enhancement by HI F may be a marker for such cells. The finding that the administration of in terferon protected mice inoculated with in terferon-resistant lymphoma cells [4] sug gested that the antitumor effect of interferon might be in part host-mediated. Our studies suggest that interferon's in vivo anticancer activity may result from its multiple biologi cal effects. HIF directly affects the tumor by retarding its growth without direct toxic ef fect. Additional means to affect the tumor results from interferon’s enhancement of the immune system to cause tumor cell elimina tion.
Acknowledgements We are grateful to Dr. J. C. Pelricciani, Dr. P. Noguchi and Dr. A. Ahmed for continuous assist ance; E. Kirshbaum and J. P. Kazanuirowski for typing and editing the manuscript.
References 1 Attallah. A. M and Strong, D. M : Differen tial effects of interferon on the MHC expres sion of human lymphocytes: enhanced expres sion of HI.A without effect on la. Int. Archs Allergy appl. Immun, (in press, 1979). 2 Attalah, A. M.; Folks, T.; Noguchi, P.: Nogu chi, C. T., and Ahmed, A.: Enhanced human natural killer and antibody-dependent cell cy totoxicity by incubation with human interfer on. Fed. Proc. (in press, 1979). 3 Callewort, D. M.; Lightbody, J. J.; Kaplan, J.; Jaroszewski, J.; Peterson, W. D., jr., and Ro senberg, J. C.: Cytotoxicity of human peri pheral lymphocytes in cell-mediated lympho-
381
lysis: antibody-dependent cell-mediated Iympholysis and natural cytotoxicity assays after mixed lymphocyte culture. J. Immun. 121: 81-85 (1978). 4 Gresser, 1.; Maury, C., and Brouty-Boye, D.: On the mechanism of the antitumor effect of interferon in mice. Nature, Lond. 239: 167-169 (1972). 5 Hallar, O. A.; Gidlund, M.; Kurnick, J. T., and Wigzell, H.: In vivo generation of mouse natu ral killer cells: role of the spleen and thymus. Scand. J. Immunol. 8: 207-213 (1978). 6 Harfast, B.; Anderson, T., and Perlmann, P.: Immunoglobulin-independent natural cytotox icity of Fc receptor-bearing human blood lym phocytes to mumps virus-infected target cells. J. Immun. 121: 755-761 (1978). 7 Herberman, R. R.; Ortaldo, J. R., and Bon nard, G. D.: Augmentation by interferon of human natural and antibody-dependent cellmediated cytotoxicity. Nature, I.ond. 277: 221-223 (1979). 8 Kail, M. A. and Koren, H. S.: Heterogeneity of human natural killer cell populations. Cell Immunol. 40: 58-68 (1978). 9 Kiessling, R. and Holter, O.: Natural killer cells in the mouse: an alternative immune sur veillance mechanism. Contemp. Top. Intmunobiol. 8: 171-201 (1978). 10 Koren, H. S.; Amos, D. B„ and Buckley, R. H.: Natural killing in immunodeficicnt pa tients. J. Immun. 121: 796-799 (1978). 11 Koren, H. S. and Williams, M. S.: Natural kill ing and antibody-dependent cellular cytotoxici ty are mediated by different mechanisms and by different cells. J. Immun. 121: 1956-1960 (1978). 12 Lotzova, E. and McCredie, K. B.: Natural kill er cells in mice and man and their possible biological significance. Cancer Immunol. Irnmunother. 4: 215-221 (1976). 13 Nelson, D. I..; Bundy, B. M., and Strober, W.: Spontaneous cell-mediated cytotoxicity by hu man peripheral blood lymphocytes in vitro. J. Immun. 119: 1401-1405 (1977). 14 Ojo, E. and Wigzell, H.: Natural killer cells may be the only cells in normal mouse lymph oid cell populations endowed with cytolytic ability for antibody coated tumor target cells. Scand. J. Immunol. 7: 297-306 (1978).
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Enhanced Human NK and ADC'C Activity by HIE
382
18 Welsh, R. M„ jr.: Mouse natural killer cells: induction specificity and function. J. Immun. 121: 1631-1635 (1978)
Received: March 17, 1979 Correspondence to: Dr. A. M. Attallah, Division of Pathology, N1H Bldg. 29, Room 521, Bethesda, MD 20205 (USA)
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15 Santoli, D.; Trinchieri, G., and Koprowski, H.: Cell-mediated cytotoxicity against virus-infect ed target cells in humans. J. Immun. 121: 532-537 (1978). 16 Sendo, F. T.; Aoki, E.; Boyse, A., and Boafo, C. K.: Natural occurrence of lymphocytes showing cytotoxic activity to BALB/c radia tion-induced leukemia RL-1 cells. J. natn. Can cer Inst. 55: 603-609 (1975). 17 Trinchieri, G.; Santoli, D., and Koprowski, H.: Spontaneous cell-mediated cytotoxicity in hu mans: role of interferon and immunoglobulin. J. Immun. 121: 1849-1855 (1978).
Attallah/Folks
Interferon Enhanced Human Natural Killer and AntibodyDependent Cell-Mediated Cytotoxic Activity A. M. AItallah and T. Folks Division of Pathology, Bureau of Biologies, FDA, and Department of Immunology, Naval Medical Research Institute, Bethesda, Md.
Introduction Natural killer (NK) or spontaneous cyto toxicity has been well studied, recognized and reviewed in man and other species [5, 9, 12, 18]. The absence of apparent antigen ic stimulation is the hallmark of this phe nomenon, indicating that specificity may not be important in immunosurveillance against tumors. Separation and identification of the effector cells which mediate NK activity and antibody-directed cell-mediated cytotoxicity (ADCC) have recently been attempted [8, 11, J3]. These studies suggest that the two
phenomena are mediated by different effec tor cell subpopulations and also by different mechanisms [11, 16]. Since the finding that some strains of mice showing high NK activity have low spontaneous tumor development [9], the search for NK enhancing products for possi ble cancer immunotherapy has received a great deal of attention [3], One promising candidate is interferon. Virally infected cells induce high levels of human interferon (HIF) from human peripheral blood lym phocytes (HPBLs) when cultured together [15, 17], The HPBLs show enhanced NK
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
Abstract. Preincubation of normal human lymphocytes with human interferon for 18-24 h at 37 °C resulted in an increase of the activity of both natural killer (NK) cells and anti body-mediated cytotoxic cells (ADCC). The human myeloid line, K-562, which is highly susceptible to NK cells, was employed. ADCC was assessed with antibody-coated chick erythrocytes as targets. NK cells and ADCC were detected in a 4-hour 5lCr release assay. The magnitude of the enhancement was proportionate to the amount of interferon used in preincubation of the effector cells. Prcincubation of tumor-target cells with interferon does not increase their susceptibility or resistance to lysis. The major effect of interferon on the cellular metabolism of the tumor-target cell is inhibition of DNA synthesis, but no direct cytotoxic effect was detected. Our findings may be important in understanding the mode of action of interferon in increasing host resistance to a variety of pathogens and tumors. This may be accomplished by inhibiting the growth of the tumor while simultaneously enhancing the natural killing mechanism for immunosurveillance.
Attallah Folks
activity toward these infected cells and are Fc-receptor-positivc and Ig-negative [6]. Some reports using human fibroblasts with anti-HLA or antivirus sera, and virus-in fected cells, have shown no enhancement of ADCC with HPBLs [15, 17]. The possibili ty has been raised that HIF may be acting not only on the effector cell, but also on the tumor cell. In the study reported here, HPBLs and tumor target cells were incubat ed with HIF and tested for enhanced NK and ADCC activities.
Materials and Methods
at the beginning to enhance effector and target cell contact. Plates were incubated for 4 h at 37 C in 5"/ C 0 2. At the end of incubation, the plates were spun at 500 g for 10 min, and 0.1 ml of the 0.2 ml total volume was counted for ra dioactivity (1185 automatic gamma counter, Searle, Chicago, 111.). ADCC Assay HPBLs (5XlOVlOO/d) were added to chrom ium-labeled CRBC (2X 10V50 «1) in triplicate in Linbro 96-well roundbottom microtiter trays. In addition, either 50 /d of anti-CRBC (1:20,000 fi nal dilution) or 50 «1 medium was added to the wells (total volume was 200 id). Plates were incu bated for 4 h at 37 C in a 5°/o C 0 2 incubator, at which time the plates were spun and 0.1 ml from each well was counted for radioactivity.
Effector Cells Human peripheral blood, which demonstrated high NK cell activity for all experiments, was ob tained from normal donors. Heparinized peripher al blood was layered on Ficoll-Hypaque and cen trifuged for 40 min at 500 g at 4 C. Mononuclear cells were removed and washed three times in RPMI-1640.
Interferon Source and Treatment Partially purified human lymphoblastoid inter feron induced in Namalva cells with a specific ac tivity of 1.25 X105 reference units/mg protein was used [1], HPBLs, 2X l06/ml RPMI-1640, were in cubated with HIF for 24 h at 37 °C in a 5°/o C 0 2 incubator. Cells were washed three times before use in the assay.
Target Cells For the NK assay, the myeloid leukemia cell, K-562, was utilized. It was grown in culture using complete RPMI-1640 media supplemented with 250 ug/ml gentamycin, 10 miM Hepes, 2 mM glu tamine and 10°/o heat-inactivated fetal bovine se rum (FBS) until time of the assay. 2X106 tumor cells were washed twice in RPMI-1640 and then resuspended in 100 «Ci/100 «1 of 5,Cr (Amersham). The tumor cells were incubated for 1 h at 37 °C in 5°/o C 0 2 and washed three times in RPMI-1640. Chicken red blood cells (CRBC) were freshly obtained for each ADCC assay, washed twice in complete RPMI-1640, and treated in the same way as the tumor cells.
Calculation of Percent Cytotoxicity Percent cytotoxicity using 5lCr release assay was determined by the following formula:
NK Assay HPBLs (5X105/100 «1) were incubated with chromium-labeled K-562 cells (1X104/100 m1) in triplicate in Linbro 96-well roundbottom micro titer trays. Medium was always complete RPMI1640. A slow spin (50 g for 1 min) was employed
% cytotoxicity = test cpm - spontaneous release cpm 100 x ---------------------------------------------------- • treeze/thaw cpm - spontaneous release cpm DNA, RNA and Protein Synthesis in the Target cells The myeloid leukemia cell, K-562, was sus pended to lX 106/ml in 0.1ml and dispersed in each well of a microtiter plate (Linbro Corp., New Haven, Conn.) in triplicate. Various doses of interferon (0.1 ml) were added to the culture. The cultures were then incubated at 37 °C in a humidi fied atmosphere of 5°/o C 0 2 in air for 48 h. Each culture received either 1 «Ci of 3H-TdR, 3H-uridine or 3H-leucine (Schwarz-Mann, Orangeburg, N.Y.) in 20 »1 of medium 16 h before harvest. Cells were harvested using the multiple automated sample harvester. The mean uptake and standard
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
378
Enhanced Human NK and ADCC Activity by HIF
error were calculated; percent inhibition was cal culated by the formula: % inhibition = mean cpm of culture containing HIF x 100. 1 mean cpm of culture containing medium
Results Effect of HIF on NK Activity Variation of Effector-to-Target Ratios. In order to determine the effect of interfer70 60
t 50 O 30
20 10 -
25:1
10:1
5:1
E :T RATIO
Fig. 1. Enhancement of NK activity by human interferon. HPBLs were incubated for 24 h with 5,000 U HIF ( • ) or for 24 h with medium alone (O). HPBLs (effector) were incubated with 51Crlabeled K-562 (target) for 4 h, and net percent NK cytotoxicity was determined. Vertical bars = + S.E.
70 5
O O
on on HBPLs, the effector-to-target (E:T) ratios were varied from 25:1 to 5:1, as seen in figure I. At all ratios, significant en hancement of NK activity was observed with the HIF-treated cells. The greatest enhance ment was seen at the 10:1 E:T ratio, with the HIF-treated cells having 50 ± 2%> cyto toxic activity, while the untreated cells showed 31 ± 2°/o. Dose Response with Varying Units of Interferon. Figure 2 depicts the effect of varying the units of interferon on NK activ ity. Maximal enhancement was observed when 600 U HIF were added to HPBLs and incubated for a 24-hour period. There was no further increase in cytotoxicity when HPBLs were treated with increasing doses of HIF, up to 5,000 U. Interferon Treatment of Target Cells. In order to determine whether the effect of HIF was on the effector or the target cell, K-562 cells were incubated with 5,000 U of HIF and tested 24 h later for a change in susceptibility to lysis by HPBLs (table I). No change in susceptibility occurred follow ing target treatment with HIF (table I). However, as little as 5 U HIF caused signif icant inhibition of DNA synthesis. RNA and protein synthesis was also inhibited but to a lesser extent in the tumor target cells (table II). Table I. Effect of human interferon on the suscep tibility of the target cell in the NK assay1
60 50 40
0
125 300 600 1,500 2p00 5p00 U N ITS OF INTERFERON
Fig. 2. Effect of varying unit doses of interfer on on NK activity. Condition was as in figure 1; E:T ratio was 50:1. Vertical bars = + S.E.
Target
Effector
% cytotoxicity ± S.E
K-562 + HIF (5,000 U) K-562
+ HPBLs + HPBLs
58 ± 2 64 ± 3
1 Target cells (K-562) were incubated for 24 h in either medium + 5,000 U of HIF or medium alone.
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
o
379
380
Tabic II. Effect of human interferon on DNA, RNA and protein synthesis of myeloid leukemia cells in vitro
HIF units per culture
% inhibition of 3H-TdR
0 5 50 500
3H-uridine 3H-leucine
_
_
_
32 37 53
2 24 30
15 7 22
Table III. Enhancement of ADCC following pre incubation of HIF with HPBLs1
Anti-CRBC
% ADCC ± S.E
1
1
+
1 +
+
1 +
Interferon
-
55 ± 1.5 26 ± 0.15 3.0 ± 0.1 0.1 ± 0.1
1 HPBLs were incubated either with or without HIF (5,000 U) for 24 h and tested for their ability to mediate ADCC against CRBC. E:T ratio =25:1. Final dilution of rabbit anti-CRBC was 1:20,000.
Enhancement in ADCC Activity Follow ing Interferon Treatment of HPBLs. HPBLs were either treated for 24 h with HIF (5,000 U) or not treated and then test ed for their ability to mediate ADCC against CRBC. As observed in table III, a 2-fold increase in ADCC was seen in the HIFtreated effector population, with no signifi cant increase in cytotoxicity between HIFtreated and untreated effector groups in the absence of anti-CRBC.
Discussion
The present study has investigated some of the effects of HIF on HPBLs, specifically
NK and ADCC activity. It was found that NK activity of HPBLs was enhanced ap proximately 70"/o following treatment with HIF [2], Kinetics of the NK response stud ied by varying the effector-to-target ratios indicated that either more killer cells were present or each killer cell was more effec tive, since enhancement was greater at low er ratios. When units of HIF were varied, a maximal concentration was seen at 600 U after which there was no further enhance ment with increasing doses; this could be due to saturation of the HIF receptor. In order to rule out HIF carry-over from preincubation with HPBLs and the subse quent direct effect on the target cell, investi gations were carried out to determine if sus ceptibility to lysis of the target had been al tered. Since no significant change in suscep tibility was noted, it was concluded that the NK activity was contributed solely by the effector cell. However, HIF did affect pri marily DNA; RNA and protein synthesis were affected to a lesser extent in the tumor target. Reports from various laboratories have indicated that ADCC against virus-infected cells is not enhanced over normal cells [15] and that ADCC is not enhanced in the pres ence of HPBLs which were preincubated with HIF [17], Conflicting reports have shown that HIF can enhance ADCC using tumor cell targets [7], Differences in the two systems may reside in the source of effector cells, the antitumor antibody, or the quanti ty and source of HIF. In either case, our findings [2] have shown a marked increase in ADCC (greater than 2-fold). The type of target used in our system was xenogeneic, which suggests that another type of effector cell mediates ADCC against xenogeneic tar gets. Such a subpopulation of ADCC effec
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/28/2018 5:37:19 AM
Altai Iah/Folks
tor cells has recently been described in the murine system [14]. Further studies are being carried out to determine if there arc subpopulations of human ADCC effector cells and if enhancement by HI F may be a marker for such cells. The finding that the administration of in terferon protected mice inoculated with in terferon-resistant lymphoma cells [4] sug gested that the antitumor effect of interferon might be in part host-mediated. Our studies suggest that interferon's in vivo anticancer activity may result from its multiple biologi cal effects. HIF directly affects the tumor by retarding its growth without direct toxic ef fect. Additional means to affect the tumor results from interferon’s enhancement of the immune system to cause tumor cell elimina tion.
Acknowledgements We are grateful to Dr. J. C. Pelricciani, Dr. P. Noguchi and Dr. A. Ahmed for continuous assist ance; E. Kirshbaum and J. P. Kazanuirowski for typing and editing the manuscript.
References 1 Attallah. A. M and Strong, D. M : Differen tial effects of interferon on the MHC expres sion of human lymphocytes: enhanced expres sion of HI.A without effect on la. Int. Archs Allergy appl. Immun, (in press, 1979). 2 Attalah, A. M.; Folks, T.; Noguchi, P.: Nogu chi, C. T., and Ahmed, A.: Enhanced human natural killer and antibody-dependent cell cy totoxicity by incubation with human interfer on. Fed. Proc. (in press, 1979). 3 Callewort, D. M.; Lightbody, J. J.; Kaplan, J.; Jaroszewski, J.; Peterson, W. D., jr., and Ro senberg, J. C.: Cytotoxicity of human peri pheral lymphocytes in cell-mediated lympho-
381
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Received: March 17, 1979 Correspondence to: Dr. A. M. Attallah, Division of Pathology, N1H Bldg. 29, Room 521, Bethesda, MD 20205 (USA)
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